The lipooligosaccharide (LOS) is an important virulence factor in the pathogenesis of gonococcal infection. (2). Our hypothesis is that an understanding of the molecular mechanisms which are responsible for the synthesis and regulation of these macromolecules will assist in the elucidation of there role in pathogenicity. The long term goal of our laboratory is the definition of the biosynthetic pathways which control the expression of Neisserial LOS synthesis. As a prelude to this effort, we have derived from gonococcal strain 1291, a series of 5 different stable LOS mutants. These mutants contain LOS whose oligosaccharides vary progressively by 1 or 2 saccharides from a deep rough mutant to the wild type LOS. In addition, we have prepared a group of monoclonal antibodies which define saccharide epitopes specific for four of these mutant LOSs, hence we have the capability to identify the products of expression of the LOS oligosaccharide synthesis genes from these mutants. During this proposal, we shall define the gene(s) which control the conserved terminal tetrasaccharide residue of gonococcal LOS. This epitope has been chemically characterized and is defined antigenically by the monoclonal antibodies 3Fll and 6B4. This epitope may play a role in gonococcal pathogenesis by molecular mimicry of human antigens since this tetrasaccharide has been demonstrated to be chemically identical to the human l erythrocyte antigen. We have demonstrated that this epitope is present on gonococcal LOS during human infection. During these studies, we will map the LOS oligosaccharide synthesis genes for the epitopes defining each of the LOS mutants and we will determine the relationship between pyocin nucleic acid and the LOS synthesis genes. This will be accomplished by the following specific aims: 1) The gene cluster responsible for the synthesis of the gonococcal LOS oligosaccharide in gonococcal strain 1291 which expresses the terminal tetrasaccharide recognized by monoclonal antibodies 3Fll and 6B4 will be identified by constructing genomic libraries and immunoscreening them with monoclonal antibodies 3Fll and 6B4. The identified clones will be mapped, the genes responsible for the synthesis of the terminal tetrasaccharides identified and their sequence determined. These sequences will be compared known sequences of glycosyltransferases. 2) Complementation studies using the nested deletions from recombinant genomic clones identified in aim 1 and monoclonal antibody epitope analysis will be used to identify the DNA fragments that contain the mutations giving rise to the gonococcal LOS mutants 1291a-e and to order the genes in the LOS synthesis cluster. 3) The relationship between pyocin nucleic acid and the gene cluster responsible for LOS oligosaccharide synthesis will be studied to determine what role, if any, this nucleic acid has in the generation of the gonococcal LOS mutants.